Production of Null Mutants in the Major Intestinal Esterase Gene (Ges-1) of the Nematode Caenorhabditis Elegans

Omnipotent suppressors decrease translational fidelity and cause misreading of nonsense codons. In the presence of the non-Mendelian factor [eta+], some alleles of previously isolated omnipotent suppressors are lethal. Thus the current search was conducted in an [eta+] strain in an effort to identify new suppressor loci. A new omnipotent suppressor, SUP39, and alleles of sup35, sup45, SUP44 and SUP46 were identified. Efficiencies of the dominant suppressors were dramatically reduced in strains that were cured of non-Mendelian factors by growth on guanidine hydrochloride. Wild-type alleles of SUP44 and SUP46 were cloned and these clones were used to facilitate the genetic analyses. SUP44 was shown to be on chromosome VII linked to cyh2, and SUP46 was clearly identified as distinct from the linked sup45.     

Defined set of cloned termination suppressors: in vivo activity of isogenetic UAG, UAA, and UGA suppressor tRNAs.

We have cloned an isogenetic set of UAG, UAA, and UGA suppressors. These include the Su7 -UAG, Su7 -UAA, and Su7 -UGA suppressors derived from base substitutions in the anticodon of Escherichia coli tRNATrp and also Su9 , a UGA suppressor derived from a base substitution in the D-arm of the same tRNA. These genes are cloned on high-copy-number plasmids under lac promoter control. The construction of the Su7 -UAG plasmid and the wild-type trpT plasmid have been previously described ( Yarus , et al., Proc. Natl. Acad. Sci. U.S.A. 77:5092-5097, 1980). Su7 -UAA ( trpT177 ) is a weak suppressor which recognizes both UAA and UAG nonsense codons and probably inserts glutamine. Su7 -UGA ( trpT176 ) is a strong UGA suppressor which may insert tryptophan. Su9 ( trpT178 ) is a moderately strong UGA suppressor which also recognizes UGG (Trp) codons, and it inserts tryptophan. The construction of these plasmids is detailed within. Data on the DNA sequences of these trpT alleles and on amino acid specificity of the suppressors are presented. The efficiency of the cloned suppressors at certain nonsense mutations has been measured and is discussed with respect to the context of these codons.     

Altered 40 S ribosomal subunits in omnipotent suppressors of yeast

The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.     

Close Linkage Between Ochre and Missense Suppressors in Escherichia coli

It was previously shown that the ochre suppressor mutation sup15B in Escherichia coli determines the accumulation of altered 30S ribosomal subunits and the presence of altered transfer ribonucleic acid (tRNA) capable of suppressing in vitro the UAG codon. This mutation has been mapped in the present study by means of conjugation and transduction experiments. After establishing the location of sup15B near argH, the following order was determined for the markers tested: metB-argH-(sup15B, supA36)-rif-thi. A comparison of location, growth rate, and suppressor pattern determined by sup15B and supM indicates the high probability that both suppressor mutations are identical. This study has also yielded a more precise location for the rifampin resistance gene. The most interesting finding is the very close (if not adjacent) location of the suppressor mutations sup15B and supA36, both of which determine tRNA alterations.     

A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors

Summary A spontaneous mutant was isolated that harbors a weak suppressing activity towards a UAG mutation, together with an inability to grow at 43° C in rich medium. The mutation is shown to be associated with an increased misreading of UAG at certain codon contexts and UAA. UGA, missense or frameshift mutations do not appear to be misread to a similar extent. The mutation gives an increased efficiency to several amber tRNA suppressors with-out increasing their ambiguity towards UAA. The ochre suppressors SuB and Su5 are stimulated in their reading of both UAG and UAA with preference for UAG. An opal suppressor is not affected. The effect of the mutation on the efficiency of amber and ochre suppressors is dependent on the codon context of the nonsense codon.The mutated gene (uar) has been mapped and found to be recessive both with respect to suppressor-enhancing ability as well as for temperature sensitivity. The phenotype is partly suppressed by the ochre suppressor SuC. It is suggested that uar codes for a protein, which is involved in translational termination at UAG and UAA stop codons.     

A yeast tRNA mutant that causes pseudohyphal growth exhibits reduced rates of CAG codon translation

In Saccharomyces cerevisiae, the SUP70 gene encodes the CAG‐decoding tRNA^Gln_CUG. A mutant allele, sup70‐65, induces pseudohyphal growth on rich medium, an inappropriate nitrogen starvation response. This mutant tRNA is also a UAG nonsense suppressor via first base wobble. To investigate the basis of the pseudohyphal phenotype, 10 novel sup70 UAG suppressor alleles were identified, defining positions in the tRNA^Gln_CUG anticodon stem that restrict first base wobble. However, none conferred pseudohyphal growth, showing altered CUG anticodon presentation cannot itself induce pseudohyphal growth. Northern blot analysis revealed the sup70‐65 tRNA^Gln_CUG is unstable, inefficiently charged, and 80% reduced in its effective concentration. A stochastic model simulation of translation predicted compromised expression of CAG‐rich ORFs in the tRNA^Gln_CUG‐depleted sup70‐65 mutant. This prediction was validated by demonstrating that luciferase expression in the mutant was 60% reduced by introducing multiple tandem CAG (but not CAA) codons into this ORF. In addition, the sup70‐65 pseudohyphal phenotype was partly complemented by overexpressing CAA‐decoding tRNA^Gln_UUG, an inefficient wobble‐decoder of CAG. We thus show that introducing codons decoded by a rare tRNA near the 5′ end of an ORF can reduce eukaryote translational expression, and that the mutant tRNA_CUG^Gln constitutive pseudohyphal differentiation phenotype correlates strongly with reduced CAG decoding efficiency.     

The genetics of a small autosomal region of Drosophila melanogaster containing the structural gene for alcohol dehydrogenase. III. Hypomorphic and hypermorphic mutations affecting the expression of hairless

A lethal locus (l(2)br7;35B6-10), near Adh on chromosome arm 2L of D. melanogaster, is identified with Plunkett's dominant suppressor of Hairless (H). Of eight new alleles, seven act as dominant suppressors of H, the eighth is a dominant enhancer of H. One of the suppressor alleles is both a leaky lethal and a weak suppressor of H. Confirming Nash (1970), deletions of l(2)br7 are dominant suppressors, and duplications are dominant enhancers of H. A simple model is proposed to account for the interaction of l(2)br7 and H, assuming that amorphic (or hypomorphic) alleles of l(2)br7 suppress H and that hypermorphic alleles enhance H.     

Omnipotent Suppressors Effective in psi Strains of SACCHAROMYCES CEREVISIAE: Recessiveness and Dominance

We have characterized recessive and dominant omnipotent suppressor mutations obtained by conversion of the leu2-1 UAA mutation and the met8-UAG mutation in a ψ⁺ strain of Saccharomyces cerevisiae. The suppressors that act recessively upon these markers fell into two complementation groups; the sup47 and sup36 suppressors show linkage to the tyr1 locus and the aro1 locus, respectively. Of the suppressors acting dominantly upon both markers, those linked to the tyr1 locus are alleles of the SUP46 ribosomal mutation. The sup47 suppressors differ from the SUP46 suppressors not only in their suppressor activities in heterozygous diploids but also in their map positions relative to the tyr1 locus and their effects on the S11 ribosomal protein. The remaining dominant suppressors are not alleles of sup36 as judged by linkage analysis. The recessive suppressors and the dominant suppressors also differ in their effects on cell growth.     

A nucleotide change in the anticodon of an Escherichia coli serine transfer RNA results in supD-amber suppression.

The tRNAs specified by the wild type and amber suppressor alleles of the Escherichia coli supD gene have been identified, and their primary structures determined. The sequences differ by a single nucleotide in the middle of the anticodon. A CUA anticodon allows the suppressor tRNA to read the UAG stop codon; the CGA anticodon in the minor serine tRNA species from which the suppressor is derived is specific for the serine codon UCG.     

Suppression of termination mutations caused by defects of the NMD machinery in Saccharomyces cerevisiae

Among a large collection of nonsense (termination) suppressors of Saccharomyces cerevisiae, a few remained obscure for their molecular nature. Of those, a group of weak and recessive suppressors, sup111, sup112 and sup113, is of particular interest because of their dependency on [PSI+], a yeast prion. From the facts that these suppressors map at positions quite similar to the UPF2, UPF3 and UPF1 genes, respectively, and that some mutations in the UPF genes confer termination suppressor activity, we suspected that sup111, sup112 and sup113 would very well be mutant alleles of the UPF genes. We tested our speculation and found that sup113, sup111 and sup112 were in fact complemented with the wild-type alleles of UPF1, UPF2 and UPF3, respectively. We further obtained evidence that the UPF1, UPF2 and UPF3 loci of the strains carrying sup113, sup111 and sup112, respectively, had point mutations. From these results, we conclude that sup111, sup112 and sup113 are mutant alleles of UPF2, UPF3 and UPF1, respectively, and thus attribute suppressor activity of these mutations to defects in the NMD (nonsense-mediated mRNA decay) machinery.     

Exceptional codon recognition by the glutamine tRNAs in Saccharomyces cerevisiae.

Recently, it was shown that wild-type glutamine tRNAs in yeast cause low-level nonsense suppression that can be enhanced by increasing glutamine tRNA gene copy number. In order to investigate glutamine tRNA behavior further, anticodon mutations that confer nonsense suppression were identified in yeast sup70 gene, which codes for glutamine tRNA(CAG). In this study we show that suppressors derived by mutation severely limit growth such that suppressor-bearing spores germinate but arrest cell division at approximately the 50 cell stage. Analysis of a sup70 deletion was used to establish that growth limitation results from loss of wild-type glutamine tRNA(CAG) function. By exploiting the growth inhibition of sup70 alleles, some exceptional codon recognition properties of glutamine tRNAs were revealed. Our results indicate that amber suppressor glutamine tRNA(UAG) can translate 5'-CAG-3' glutamine codons with low efficiency in the presence of an A/C mismatch at the first position of the codon, suggesting that reading may occur at a low level by a two-out-of-three reading mechanism. In addition, when glutamine tRNA(CAA) is over-expressed in vivo, it translates 5'-CAG-3' codons using a mechanism that resembles prokaryotic-like U/G wobble, which normally does not occur in yeast. Our studies also suggest that the yeast glutamine tRNA suppressors could potentially be exploited to express ciliated protozoan genes that normally contain internal 5'-UAG-3' and 5'-UAA-3' codons.     

Mutational alterations of tryptophan-specific transfer RNA that generate translation suppressors of the UAA, UAG and UGA nonsense codons

The recessive lethal amber suppressor su⁺7(UAG-1) in Escherichia coli inserts glutamine in response to the UAG codon. The genetic analysis presented in this paper shows that the su^?7 precursor allele can give rise to suppressors of the UGA codon as well as of the UAG codon. This observation suggests that the su^?7 gene normally codes for transfer RNA^Trp, a tRNA whose anticodon can be modified by single base changes to forms that can translate either UAG or UGA. The chemical findings presented in the accompanying paper (Yaniv et al., 1974) are wholly in accord with this interpretation. Thus, a single base substitution in the anticodon sequence of a tRNA can affect both the coding specificity of the molecule and also the amino acid acceptor specificity.     

Novel nematode amber suppressors

Nine amber suppressor mutations were isolated in the nematode Caenorhabditis elegans by reverting amber alleles of a sex-determining gene, tra-3. One suppressor maps to a known locus, sup-5 III , but the other eight map to three new loci, sup-21 X (five alleles), sup-22 IV (two alleles) and sup-23 IV (one allele). Amber alleles of tra-3 and of a dumpy gene, dpy-20, were used to measure the efficiency of suppression; the sup-21 and the sup-22 alleles were both shown to be heterogeneous and generally weaker suppressors than sup-5 alleles, which are homogeneous. The spectrum of mutations suppressed by a strong sup-21 allele, e1957, was investigated and compared to the spectra for the amber suppressors sup-5 III and sup-7 X, using amber alleles in 13 assorted genes. Some of the differences between these spectra may be due to limited tissue specificity in sup-21 expression.—Suppression of dpy-20 was used to show that the sex-linked suppressors sup-7 and sup-21 are not dosage compensated in male (XO) relative to hermaphrodite (XX).—Several uses of amber suppressors are critically discussed: for identifying null mutations, for varying levels of gene activity and for detecting maternal mRNA.     

Neurospora crassa supersuppressor mutants are amber codon-specific

Neurospora crassa has 10 mapped supersuppressor (ssu) genes. In vivo studies indicate that they suppress amber (UAG) premature termination mutations but the spectrum of their functions remains to be elucidated. We examined seven ssu strains (ssu-1, -2, -3, -4, -5, -9, and -10) using cell-free translation extracts. We tested suppression by requiring it to produce firefly luciferase from a reading frame containing premature UAA, UGA, or UAG terminators. All mutants except ssu-3 suppressed UAG codons. Maximal UAG suppression ranged from 15% to 30% relative to controls containing sense codons at the corresponding position. Production from constructs containing UAA or UGA was 1-2%, similar to levels observed with all nonsense codons in wild-type and ssu-3 extracts. UAG suppression was also seen using [35S]Met to radiolabel polypeptides. Suppression enabled ribosomes to continue translation elongation as determined using the toeprint assay. tRNA from supersuppressors showed suppressor activity when added to wild-type extracts. Thus, these supersuppressors produce amber suppressor tRNA.     

Prion-Dependent Lethality of sup45 Mutants in Saccharomyces cerevisiae

In yeast Saccharomyces cerevisiae translation termination factors eRF1 (Sup45) and eRF3 (Sup35) are encoded by the essential genes SUP45 and SUP35 respectively. Heritable aggregation of Sup35 results in formation of the yeast prion [PSI⁺]. It is known that combination of [PSI⁺] with some mutant alleles of the SUP35 or SUP45 genes in one and the same haploid yeast cell causes synthetic lethality. In this study, we perform detailed analysis of synthetic lethality between various sup45 nonsense and missense mutations on one hand, and different variants of [PSI⁺] on the other hand. Synthetic lethality with sup45 mutations was detected for [PSI⁺] variants of different stringencies. Moreover, we demonstrate for the first time that in some combinations, synthetic lethality is dominant and occurs at the postzygotic stage after only a few cell divisions. The tRNA suppressor SUQ5 counteracts the prion-dependent lethality of the nonsense alleles but not of the missense alleles of SUP45, indicating that the lethal effect is due to the depletion of Sup45. Synthetic lethality is also suppressed in the presence of the C-proximal fragment of Sup35 (Sup35C) that lacks the prion domain and cannot be included into the prion aggregates. Remarkably, the production of Sup35C in a sup45 mutant strain is also accompanied by an increase in the Sup45 levels, suggesting that translationally active Sup35 up-regulates Sup45 or protects it from degradation.Key Words: Sup45, Sup35, eRF1, eRF3, amyloid, [PSI⁺], translation termination, Saccharomyces cerevisiae     

Selective systems for obtaining recessive ribosomal suppressors in saccharomycete yeasts

Recessive mutations only occurring in two genes (ribosomal suppressors sup1 and sup2) can be obtained using special selective system. We demonstrate that the absolute selectivity of the system is based on selection for simultaneous reversions to prototrophy in mutants requiring adenine and histidine in haploids marked by two different nonsense mutations--his7-1 (UAA) and ade1-14 (UGA, this being identified in the present study). In support to this conclusion, we developed an analogous system utilising his7-1 (UAA) and lys2-87 (UGA). The selectivity of the system is shown to be influenced both by the choice of nonsense alleles and by genotypic background.     

An amber mutation in the gene encoding the beta'' subunit of Escherichia coli RNA polymerase.

An Escherichia coli strain carrying an amber mutation (UAG) in rpoC, the gene encoding the beta prime subunit of RNA polymerase, was isolated after mutagenesis with nitrosoguanidine. The mutation was moved into an unmutagenized strain carrying the supD43,74 allele, which encodes a temperature-sensitive su1 amber suppressor, and sue alleles, which enhance the efficiency of the suppressor. In this background, beta prime is not synthesized at high temperature. Suppression of the mutation by the non-temperature-sensitive amber suppressor su1+ yields a protein which is functional at all temperatures examined (30, 37, and 42 degrees C).